1. Field of the Invention
The present invention relates to a composition and method for producing an optimum ovulation response in mammals.
2. Description of the Prior Art
In mammals, the fetal or neonatal female produces thousands of oocytes, most of which are never ovulated and most of those ovulated are never fertilized. A multimillion dollar industry has developed that is concerned with methods to ovulate, fertilize and utilize these embryos either in the mother or by transfer of these oocytes to surrogate mothers. The advantages of such procedures include increasing the reproductive rate of valuable mammals, by decreasing the generation interval; enabling the progeny testing of females, the use of superior females as donors, and increasing the number of progeny per female through controlled multiple births, and transporting embryos with selected genetic characteristics to distant places.
It is also possible and especially desirable to stimulate the production of ova at each estrus, effectively multiplying the reproductive capacity of the mammal. The introduction of multiple follicle growth with subsequent ovulation is referred to as "superovulation" and promotes out of season breeding and twinning. For example, it is an established commercial practice to stimulate the formation of multiple ovarian follicles in cattle and other domestic animals by multiple injections of follicle stimulation hormone.
It is an established commercial practice to stimulate estrus and ovulation in sheep, rabbits and other mammals at a time other than their normal breeding season or period. It is also a commercial husbandry practice to induce twinning in cattle and increase the numbers of offspring in sheep, pigs, and other species.
The all important first step in these procedures is to produce a superovulation response in a superior female donor. The objective of superovulation is to increase the number of normal fertile eggs or embryos per donor. The basic principle of superovulation is to stimulate extensive follicular development through intramuscular or subcutaneous administration of a preparation having follicle-stimulating hormone (FSH) activity at levels in excess of normal endogenous levels. The most commonly used sources for this preparation are swine pituitary extracts or pregnant mares' serum. The gonadotropic hormones have become known as follicle-stimulating hormone (FSH) and luteinizing hormone (LH) based on their effects on ovarian follicular activity. The activity of FSH and LH preparations are usually measured by bioassay and compared to a reference standard. Typical reference standards are the National Institute of Health standards which are designated NIH-FSH-S1 and NIH-LH-S1 for FSH and LH respectively. The S1 refers to the NIH standard #1.
Treatment of mammals, for example, cattle, horses, rabbits and many exotic (wild) species with gonadotropins leads to ovulation of numerous ova instead of the usual one. In cattle, gonadotropin treatment is usually initiated between days 9 and 14 of the estrus cycle (estrus is day 0), causing ovarian follicles to grow. Two or three days after the start of treatment, prostaglandin F.sub.2a or an analog is injected to terminate the luteal phase of the estrus cycle prematurely by lysing the corpus luteum; about 2 days later estrus occurs. Estrus lasts about 12 to 18 hours and ovulation occurs about 28 hours after the onset of estrus, and fertilization probably occurs a few hours after ovulation.
Before prostaglandins became available, superovulation was initiated about 4 to 5 days before the end of the estrus cycle, a time that could not be estimated accurately. Availability of prostaglandin F.sub.2a has improved the efficacy of superovulation and has also provided flexibility in scheduling donors.
Because the best bulls are usually propagated only with frozen semen, artificial insemination is used routinely for valuable cows. Sometimes mixtures of semen from two or three bulls are used with superovulation, and the progeny are sorted out after birth on the basis of blood type.
Bovine embryos usually move from the oviduct to the uterus 72 hours after the onset of estrus, although in superovulated cows a few remain in the oviduct through day 7. A high percentage of embryos can usually be recovered nonsurgically from the uterus six or seven days after the beginning of estrus. Recovery of embryos from the oviduct requires surgery and, therefore, is recommended only in certain cases of infertility.
To recover embryos, a Foley catheter is inserted through the cervix into the uterus by palpating through the wall of the rectum with one hand as is done for artificial insemination. The latex catheter consists of three channels for inflow, outflow, and inflation of a balloon-like cuff that prevents the escape of fluid after insertion. Each uterine horn is filled and emptied five to ten times with 30 to 200 milliliters of fluid each time, according to the size of the uterus. The embryos are flushed out with this fluid into large graduated cylinders. Embryos can be filtered or allowed to settle for 30 minutes and can then be located under a stereomicroscope by searching through an aliquot from the bottom of the cylinder. They are then stored in small containers until transfer.
Embryos and ova from the one-cell to the early blastocyst stage (7 to 8 days after estrus) are between 120 and 140 micrometers in diameter exclusive of the zona pellucida. Between days 8 and 10, they double in diameter, hatch from the zona pellucida, and then grow to 20 centimeters or more in length by day 18. Since bovine embryos form no intimate attachment to the uterus before day 18, they can be recovered nonsurgically until this time, although they are increasingly prone to damage after day 14. It appears that a larger number of normal embryos can be obtained non-surgically 6 to 8 days after estrus than at other times.
It has been shown (Donaldson et al., Theriogenology 23, 189 (1985); Donaldson et al., Theriogenology 25, 749 (1986)), that luteinizing hormone (LH) contamination of follicle stimulating hormone (FSH) reduces the superovulation response in cattle. The excessive variability in superovulation response in cattle to a standardized quantity of FSH was reported in 1944 (Hammond et al., Journal Agricultural Science 34, 1 (1944)), but it was not until forty years later when the dynamics of follicular development and the response to exogenous gonadotropins was described (Monneaux et al., Theriogenology 19, 55 (1983); Moor et al., Theriogenology 21, 103 (1984)) that more reliable superovulation techniques began to be developed. It has been shown that commercial FSH preparations have high and variable LH contents (Murphy et al., Theriogenology 21, 117 (1984); Lindsell et al., Theriogenology 25, 167 (1986)). Excess LH in a superovulatory hormone has been shown to cause premature stimulation of the oocyte (Moor et al. Theriogenology 21, 103 (1984)). Rat oocytes produced by superovulation have been shown to exhibit reduced fertilization rates (Walton et al., Journal of Reproduction and Fertility 67, 91 (1983); Walton et al., Journal of Reproduction and Fertility 67,309 (1983)). Low fertilization rates in superovulated cattle have been shown not to have resulted from the quantity of semen used or the number of times the cow was bred (Donaldson, Veterinary Record 117, 35 (1985)).
It has been shown that normal preovulatory progesterone (P4) LH and FSH concentrations are necessary for optimal embryo production from superovulated cows (Donaldson, Theriogenology 23, 441 (1985); Calleson et al., Theriogenology 25, 71 (1986)). Abnormal concentrations of P4, LH and FSH are followed by abnormal follicular/oocyte maturation and lowered embryo production.
A commonly available FSH preparation manufactured by Armour Pharmaceutical Co. and known as FSH-P is a crude pituitary extract having a high and variable LH content. The LH content has been measured and the FSH/LH ratio has been found to be less than 100. Armour Pharmaceutical Co. is the assignee of U.S. Pat. Nos. 2,799,621 and 3,119,740 which relate to the preparation of FSH-P.
U.S. Pat. No. 2,799,621 to Steelman is directed to a method for recovering both adrenocorticotropin (ACTH) and gonadotropins (FSH and LH) from the same batch of pituitary material.
U.S. Pat. No. 3,119,740 to Steelman, et al. is directed to a method for preparing follicle stimulating hormone (FSH) free from contaminant physiological factors.
Development of reliable superovulation methods in cattle for producing adequate and predictable numbers of embryos has been slow (Moor et al., Theriogenology 21:103-116 (1984)). As noted above, the excessive variability in the numbers of ova shed in response to a standardized amount of injected hormone was first reported in 1944 (Hammond et al., Journal Agricultural Science 34, 1 (1944)), but it was not until 1983 (Monneaux et al., Theriogenology 19, 55 (1983); Moor et al., Theriogenology 21:103-116 (1984)) that the reasons for this variability began to be understood. The dynamics of follicular development during the bovine estrus cycle, the response to exogenous gonadotropins (Moor et al., Theriogenology 19, 55 (1983); Moor et al., Theriogenology 21:103-116 (1984)), and the differences in the relative abundance of FSH and LH activity in gonadotropin preparations (Murphy et al., Theriogenology 21:117-125 (1984)) contribute to this variability. The ratio of FSH to LH activity in the various hormone preparations used for superovulation varies between batches of Armour's FSH-P and between FSH-P and pregnant mare serum gonadotropin (PMSG) (Monneaux et al., Theriogenology 19:55-64 (1983); Murphy et al., Theriogenology 22:205-212 (1984)). FSH stimulates the growth of granulosa cells in preantral and small antral follicles (Monneaux et al., Theriogenology 19:55-64 (1983)) and reverses the process of atresia in follicles over 1.7 mm in diameter (Moor et al., Theriogenology 21:103-116 (1984)). The LH surge is responsible for the resumption of meiosis in the preovulatory oocyte, and the reduction in the high LH content of pituitary gonadotropin preparations should decrease premature activation of oocytes during superovulation (Moor et al., Theriogenology 21:103-116 (1984)). A previous study (Donaldson, Theriogenology 22:205-212 (1984)) showed that embryo production depended upon the dose of FSH-P. As the dose increased above an optimal 28 mg, three embryo production endpoints declined: the number of transferable embryos, the total embryos recovered, and the percent transferable. The number of collections at which no embryos were recovered also increased.
Considering the potential immunological reactions that might be encountered, employing bovine preparations in treatments involving cattle seems appropriate. The purification of bovine FSH has been reported (Beckers et al., Biochemie 59:825-831 (1977); Cheng, Biochem. J. 159:651-659 (1976); Grimek et al. Endocrinology 104:140-147 (1979)). However, the content of FSH in bovine pituituaries is relatively low and the recovery with purification is generally poor. Porcine pituitaries are as readily available and the FSH content seems more amenable to extraction and processing. Indeed, commercially available preparations of porcine origin have been widely used in veterinary medicine. Methods for the purification of porcine FSH have also been described (Closset et al., Eur. J. Biochem. 86:105-113 (1978); Whitley et al., Endocrinology 102:1874-1886 (1978)). The amino acid sequence for porcine FSH has been proposed (Closet., Eur J. Biochem. 86:115-120 (1978)), but there is no reported sequence for the bovine hormone.
It has been shown how LH is needed in all mammals to act on follicular cells to produce estrogen in developing follicle, i.e., a prerequisite to stimulation by FSH in superovulation (JoAnne S. Richards, Physiological Review, Vol. 60, No. 1, January 1980, pp. 51-89). The Richards publication directed investigators to consider adding LH to available FSH in order to try to improve superovulation. In the mammal species, including goats, sheep, and laboratory animals, it was reported that same problem regarding superovulated mammals with FSH and PMSG (pregnant mare serum gonadotrophin) resulted as was reported in the cattle species, i.e., fertilization failure and degeneration of embryos. The investigators reported "FSH-P was found more consistently to result in increased ovulation rate with a lower incident of excess large follicles which failed to ovulate than PMSG". The investigators did not realize that this enhanced results with FSH-P was due to the LH content and effect. (D. T. Armstrong, et al, Theriogenology, January, 1983, Vol. 19, No. 1, pp. 30-42). Continuing investigations determined that studies of the effectiveness of FSH-P, when administered at an approximate dose in combination with progestagen to induce estrus and fertile mating in seasonally anestrous ewes; however, no advantage over PMSG was evident and a major disadvantage of the FSH was the need to administer the FSH in multiple doses because of its short half-life (D. T. Armstrong, et al, Proc. 10th International Congress Animal Reproduction ART. INSEM., Urbana, Ill., USA, June 10-14, 1984, pp. VII-VIII-VII-XV).
In 1983, a paper entitled Factors Regulating Growth of Preovulatory Follicle in the Sheep and Human was presented by D. T. Baird and published in Journal Reproduction and Fertilization (1983), 69, 343-352, the similarity between sheep and humans in relying on LH and FSH to cause growth in follicles was presented. Further investigation by Scott C. Chappel, et al, published in Endocrine Reviews 4[2] 179-211 (1987) concluded that early follicular phase requires FSH and LH, with LH causing production of androgenic steroids and FSH utilizing these steroids and connects these steroids to estrogens. Follicle growth as a result of local estrogen and FSH stimulation is a result. It would appear from these and other papers that enhancement of ovulation requires some LH in the beginning and FSH to continue the process in all mammals.
Earlier investigations established that follicular development (growth) is fundamentally the same in all mammalian species. Investigators are interested in the growth of preantral follicles and this requires LH to cause the production of estrogens which, with FSH, cause follicles to grow (Scott C. Chappel, et al and JoAnne S. Richards). These earlier investigations, recognizing the need for LH resulted in many investigators adding LH to available FSH which had LH present as unknown contamination, thus resulting in confusing as well as conflicting data.